Methods and systems for determining haplotypes and phasing of haplotypes

ABSTRACT

The present disclosure provides methods and systems for determining and/or characterizing one or more haplotypes and/or phasing of haplotypes in a nucleic acid sample. In particular, the disclosure provides methods for determining a haplotype and/or phasing of haplotypes in a nucleic acid sample by incorporating synthetic polymorphisms into fragments of a nucleic acid sample and utilizing the synthetic polymorphisms in determining one or more haplotypes and/or phasing of haplotypes.

The present application claims priority to U.S. Provisional patentapplication Ser. No. 61/673,052 filed Jul. 18, 2012, which isincorporated herein by reference in its entirety.

BACKGROUND

The efforts of the Human Genome Project opened a broader window to thehuman genome. The work to further unlock the human genome is ongoing.The HapMap (Haplotype Map) Project is a global scientific effortdirected at discovering genetic variants that lead to disease bycomparing genomic information from people without a particular diseaseto those with that disease. Alleles, one or more forms of a DNA sequencefor a particular gene, can contain one or more different geneticvariants. Identifying haplotypes, or combinations of alleles atdifferent locations, or loci, on a particular chromosome is a main focusof the HapMap Project. Identified haplotypes where the two groups differmight correlate to locations of genetic anomalies that cause disease. Assuch, HapMap results will help to describe the common patterns ofgenetic variation in humans and whether those variations are potentiallycorrelated to disease. Research efforts in determining haplotypes willhelp illuminate the common patterns of genetic variation in humans andwhether those variations are potentially correlated to a particulardisease. Indeed, many researchers agree that haplotyping a genome willbe advantageous, if not essential, in relating genetic variation tophenotype and disease. Further, a particular haplotype may be correlatedto the success or failure of a treatment regimen and as such could beuseful in helping a clinician decide on a therapeutic regimen for aparticular individual that might have the highest degree of success indisease eradication in that individual.

However, there are many technical challenges associated with genomichaplotyping. For example, next generation sequencing technologies whileincreasing the capacity and accuracy of sequencing efforts, in manycases result in short sequence reads, for example several commercialplatforms currently output per fragment reads that are less than 400nucleotides long. If two or more genetic variants located on achromosome are further apart than the sequence read length, even if thatread length is thousands of base pairs long, it may be difficult if notimpossible to define a haplotype. As such, what are needed are methodsand compositions that allow for haplotyping, in particular for geneticvariants that are farther apart on a chromosome than the sequencedlength of a piece of DNA upon which they are found.

BRIEF SUMMARY

Sequencing technologies associated with next generation sequencing canresult in short sequence reads thereby making it difficult to determinethe haplotype phasing of a genome when the sequences of interest arelocated far enough apart on the chromosome such that they are outsidethe window provided by the length of the sequence read.

The present disclosure provides methods and compositions for haplotypinggenomic samples and/or determining the phasing of haplotypes usingsynthetic polymorphisms incorporated into nucleic acids. As describedherein, nucleic acid fragments can be modified to convert nativenucleotides to synthetic or artificial polymorphisms, such as singlenucleotide polymorphisms (SNPs), or other genetic anomalies therebyproducing a pattern of engineered polymorphisms in the nucleic acidfragments to be sequenced. After sequencing, the pattern of syntheticpolymorphisms can be aligned among the fragments and the haplotype canbe determined as a result of the alignment (e.g. haplotype content orphase can be determined). In this manner, a population of modifiedfragments derived from a genomic sample can be haplotyped even if thealleles for haplotyping lie on different genomic fragments.

Methods and compositions provided herein for creating artificialpolymorphisms in a nucleic acid sequence find particular utility forhaplotype determination and characterization and/or haplotype phasing;however they can also be advantageous for other purposes. For example,the methods described herein could also be used to facilitate de novosequence assembly. Further, repeat regions that are nearly identical,for example repeated nucleotide regions such as short tandem repeats,intermediate tandem repeats, etc. as used for forensic DNAfingerprinting could be distinguished from one another by a uniquepattern of artificially introduced polymorphisms and thus a moreaccurate sequence assembly achieved. For example, for forensicsequencing determining the length of a nucleotide repeated region, theorder of intermixed repeat regions, and/or the number of repeats (i.e.,short tandem repeats, intermediate tandem repeats, etc.) can beperformed using the methods herein if the repeated regions aresufficiently long such that they cannot be fully sequenced in a single,or a paired end, sequence read.

Practicing methods disclosed herein for haplotype determination and/orhaplotype phasing, de novo sequencing, forensic purposes, etc. canprovide critical information useful for, for example, disease andtherapeutic regimen correlation. In particular, haplotypes and theirphase determinations may become critical in personalized medicine wherean individual's haplotype may not only be correlated to a disease, butmay also correlate to treatment regimen success and the like for aparticular individual.

In one embodiment, the present disclosure provides methods fordetermining the sequence of a nucleic acid sample comprising providing aplurality of nucleic acid fragments of a first length modified tocomprise a plurality of synthetic polymorphisms, preparing a nucleicacid library comprising a second plurality of fragments of nucleic acidsof a second length less than that of the first length of fragments fromsaid first plurality of nucleic acid fragments comprising a plurality ofsynthetic polymorphisms, sequencing said nucleic acid library, andaligning the plurality of synthetic polymorphisms among the sequencedfragments to determine the sequence of the nucleic acid sample based onsaid alignment. In some instances, the synthetic polymorphisms are aplurality of modified nucleotides that replace the native nucleotides ata particular location and the modified nucleotides are selected from thegroup consisting of 8-oxoguanine, dPTP, isocytosine and isoguanine. Inother instances, modifications to the nucleic acids comprise partial andincomplete bisulfite conversion of cytosines in said plurality ofnucleic acid fragments. In some instances, the synthetic polymorphismalignment comprises matching (i.e. by a computer implemented method) apattern of synthetic polymorphisms in a first nucleic acid fragmentsequence with a like pattern of synthetic polymorphisms in a secondnucleic acid fragment sequence and repeating said matching with aplurality of nucleic acid fragment sequences thereby creating a sequencealignment based on the plurality of synthetic polymorphisms in aplurality of nucleic acid fragments. In some instances, a nucleic acidlibrary is sequenced using a method selected from the group consistingof sequence by synthesis, sequence by hybridization, sequence byligation, single molecule sequencing, nanopore sequencing,pyrosequencing and polymerase chain reaction. In some instances, asequence is determined by fluorescence detection. In preferredinstances, the determined sequence comprises one or more haplotypes andfurther comprises determining the phase of two or more haplotypes in thenucleic acid sample. Oftentimes, the haplotypes for phasing are locatedon different sequenced fragments. The above disclosed methods could alsobe used for de novo sequencing.

In another embodiment, the present application discloses a method forcharacterizing one or more haplotypes of a nucleic acid samplecomprising providing a pool of fragmented nucleic acids, introducing aplurality of synthetic polymorphisms such as single nucleotidepolymorphisms in the fragmented nucleic acids of said pool to producefragments comprising a plurality of synthetic polymorphisms, preparing alibrary of nucleic acid fragments that are shorter in length than theoriginal pool of fragments comprising a plurality of modified nucleicacids, sequencing nucleic acid fragments in the library, aligning thesynthetic polymorphisms of the sequenced nucleic acid fragments, andcharacterizing one or more haplotypes of the nucleic sample from thealigned synthetic polymorphisms of the sequenced fragments. In someinstances, the plurality of synthetic single nucleotide polymorphismsreplaces the native nucleotides at the site of incorporation andcomprises a plurality of modified nucleotides. In some instances, themodified nucleotides are selected from the group consisting of8-oxoguanine, isocytosine, isoguanine and dPTP. In some instancesintroduction of the synthetic polymorphisms is accomplished by partialand incomplete bisulfite conversion of cytosines in the nucleic acidfragments. In some instances, the synthetic polymorphisms are aligned bymatching (i.e., by a computer implemented program) a pattern ofsynthetic polymorphisms in a first nucleic acid fragment sequence with alike pattern of synthetic polymorphisms in a second nucleic acidfragment sequence and repeating said matching in a plurality of nucleicacid fragment sequences thereby creating a sequence alignment from thesynthetic polymorphisms in the sequenced nucleic acid fragments. In someinstances, sequencing is performed by one of sequence by synthesis,sequence by hybridization, sequence by ligation, single moleculesequencing, nanopore sequencing, pyrosequencing and polymerase chainreaction methodologies. In some instances, sequences are determined byfluorescence detection. In some instances, sequences are used todetermine the phase of two or more haplotypes in the nucleic acidsample. Oftentimes, the haplotypes for phasing are located on differentsequenced fragments. In other instances, the method described above canbe used for de novo sequencing.

In another embodiment, the present disclosure describes a method foridentifying one or more haplotypes of a nucleic acid sample comprisingproviding a nucleic acid molecule having a plurality of nucleotides,modifying a plurality of the nucleotides in the nucleic acid molecule,thereby producing a modified nucleic acid molecule comprising naturaland modified nucleotides, amplifying the modified nucleic acid moleculeto produce a plurality of modified nucleic acid copies of a firstlength, fragmenting the amplified modified nucleic acid copies underconditions to produce a library of nucleic acid fragments of a secondlength, wherein individual nucleic acid fragments in the library have aregion of sequence overlap with at least one other nucleic acid fragmentin the library and wherein the region of sequence overlap comprises atleast one modified nucleotide, determining the sequence of nucleic acidfragments of the library, and aligning the sequence of nucleic acidfragments by the locations of the modified nucleotides in the regions ofsequence overlap to identify one or more haplotypes of the nucleic acidmolecule. In some instances, the nucleic acid molecule comprises severaldifferent nucleotide types along the length of sequence and one of thenucleotide types may be modified in the modified nucleic acid or all ofthe nucleotides of the one type may be modified in the modified nucleicacid. In some instances, only a subset of the nucleotides of the onetype is modified in the modified nucleic acid. In some instances,methods for identifying a haplotype further comprises determining thephase for at least two haplotypes in the nucleic acid molecule.Oftentimes the haplotypes for phasing are located on different sequencedfragments. In some instances for haplotyping, the nucleic acid moleculecomprises several different nucleotide types along the length ofsequence, wherein the at least two haplotypes are bi-allelic for two ofthe nucleotide types, and wherein a third nucleotide type is modified inthe modified nucleic acid. In other instances, at least two haplotypesare bi-allelic for nucleotide types that are selected from the groupconsisting of A, T and G, and wherein C is modified to U in the modifiednucleic acid. In other instances, at least two haplotypes are bi-allelicfor T and G, and wherein C is modified to U in the modified nucleicacid. In additional embodiments, at least two haplotypes are bi-allelicfor nucleotide types that are selected from the group consisting of A, Tand C, and wherein G is modified to 8-oxo-G in the modified nucleicacid. In other instances, at least two haplotypes are bi-allelic for Cand T, and further G is modified to 8-oxo-G in the modified nucleicacid.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment for incorporating the modified nucleotide8-oxoguanine (8-oxo G) into DNA thereby converting natural nucleotidesin a sequence to synthetic polymorphisms in a sequence.

FIG. 2 shows an embodiment for incorporating synthetic polymorphismsinto a polynucleotide by partial sodium bisulfite conversion ofcytosines to uracils in DNA.

FIG. 3 depicts an embodiment for incorporating synthetic polymorphismsinto a polynucleotide by incorporating the modified nucleotidesisocytosine and isoguanine into DNA in lieu of the native nucleotides.

FIG. 4 demonstrates an embodiment where the target DNA containsartificial polymorphisms created using sodium bisulfite conversionmethodology. The natural occurring SNPs (bolded and enlarged) on A)allele 1 G and C and B) allele 2 T and A are separated by a distancegreater than a typical insert library size and therefore the SNP phasingis indeterminable, whereas artificial C to T polymorphisms which can beincorporated in the nucleic acid by partial bisulfite conversion can beused to align sequenced fragments so that a haplotype can be determinedfor the two alleles.

FIG. 5 shows an example of haplotype reconstruction. The incorporatedartificial SNPs are depicted as vertical lines on the linear DNAfragments Allele 1 and Allele 2. The DNA is fragmented, sequenced andthe sequencing reads are aligned based on the unique pattern of theincorporated synthetic SNPs (allele 2 from FIG. 4 depicted in thisFigure). The alignment of the artificial SNPs in the overlappingfragments allows for the rebuilding of the original genomic fragmentsequence and the reconstruction of the haplotype for allele 2 can bedetermined.

FIG. 6 shows an example of how the embodiment for a “first strandextension reaction” can be used to incorporate synthetic polymorphismsinto a DNA target.

FIG. 7 shows sequencing data for the percent of modified nucleotides (%error rates) incorporated into phiX template DNA extension products forflowcell lanes 1, 2, 3 and 4.

FIG. 8 shows sequencing data for the percent of phiX sequencing reads bycycle having 0, <1, <2, <3 or <4 incorporated modified nucleotides. Yaxis is % reads with X errors or less 0-100%, X axis is cycle number0-100.

FIG. 9 shows a composite of the types and frequency (error rate) ofsynthetic polymorphisms that were introduced into the phiX template DNAfor each flowcell lane during first strand extension.

FIG. 10 A-C are representative of the distribution or coverage ofartificial polymorphisms introduced into a phiX template DNA.

FIG. 11 shows coverage plots representing the sequencing data of threeclones Panel A) Clone A, Panel B) Clone B and Panel C) Clone D. Thegraphs represent the coverage and locations of synthetic and naturalheterozygous SNPs incorporated into p53 gene sequences derived from theDNA of a Yoruban male (NA18507). Each graph reports the sequence in theapproximate same region of the p53 gene for each clone and the starsmark the approximate locations of natural heterozygous SNPs among therandomly distributed introduced synthetic SNPs. The top horizontal linewith peaks represents the reference calls and the continuous baselinewith vertical peaks under the horizontal line represents thenon-reference calls.

DETAILED DESCRIPTION

The ability to determine groups of closely linked alleles in a genomethat are inherited together, or haplotyping, may help to map humandisease genes. Disease maps could be used to diagnose, prognose and/oridentify disease or risk of disease for a patient as well as determinepotential treatment therapies unique to any one person. Such is one ofthe goals of personalized healthcare. However, the same holds true forplant and animal species, for example economically relevant plant andanimal species, wherein sequence knowledge such as haplotyping couldalso be used to advantage in veterinary and plant sciences. As such,determining a haplotype and/or phasing of haplotypes is important fromboth a biological and clinical point of view. Sequencing a sampleprovides sequence information with which an investigator can start tounravel and determine such correlations.

As used herein, the term “haplotype” refers to a haploid genotype, acombination or set of alleles or DNA sequences found at differentlocations or loci on a chromosome which are typically inherited as aunit and are linked, for example during a recombination event. Ahaplotype can provide a distinctive genetic pattern of an individual. Ahaplotype can be determined for one locus, several loci, over a portionof or for an entire chromosome. The term “allele” is used consistentwith its meaning in the art of biology. An allele is one or morealternative forms of a gene, genetic sequence or single nucleotide (e.g.a single nucleotide polymorphism or SNP) found at a specific location,or locus, on a chromosome. The term “locus” is used consistent with itsmeaning in the art of biology. A locus (plural “loci”) refers to aspecific location or place on a chromosome identified with a gene,genetic sequence or single nucleotide. As such, one or more alleles fora particular gene, for example, can be found at a particular locus on achromosome. Different genes can be identified with different loci on achromosome, wherein each gene, for example, may be associated with oneor more different allelic sequences. Alleles are not limited to anyspecific type and may include, for example, normal genetic sequences orvariant genetic sequences. For example, single nucleotide polymorphisms(SNPs), short tandem repeats (STRs), etc. can be included as variantsand genetic sequences. The term “phased alleles” refers to thedistribution of the particular alleles on a chromosome. Accordingly, the“phase” of two alleles can refer to a characterization or determinationof whether the alleles are located on a single chromosome or twoseparate chromosomes (e.g. a maternally or paternally inheritedchromosome).

Even though sequencing technologies can produce a very large number ofsequence reads, the read lengths can be relatively short. Whilenext-generation sequencing technology may increase the accuracy ofsequencing and may be useful for calling variants, the technology can beof limited use when phase, or haplotype information, is desired. Phasinginformation derived from short sequence reads have previously been verydifficult to determine unless the two polymorphisms of interest were soclose to one another that they were present on the same sequencedfragment of DNA, or perhaps in a case where one polymorphism wasdetermined to be present from a first sequence read and the secondpolymorphism was detected in the second sequence read of the same pairof nucleic acid fragments. Instances resulting from the second case arecontemplated to be rare since, on average, the human genome has onepolymorphism for every 1000 nucleotides. As such, the probability of aparticular read containing a polymorphism may be approximately 15%(sequence read length/polymorphism frequency of one polymorphism every1000 nucleotides). The combined probability of both reads belonging to apair of sequences having each one polymorphism is the product of theindividual probabilities (15%×15%). Therefore, it is contemplated that asmall subset of fragment read pairs, for example approximately 2.25% ofshort fragment read pairs, could contain two variant sequences that forma haplotype. This is further complicated when taking into account thatthe average insert size distribution of the typical sequencing library,for example a library created for a next generation sequencingtechnology can range from approximately <50 bp (e.g., Life TechnologiesSOLiD sequencing at mate paired sequencing) to approximately <400 bp(e.g., 454 Life Sciences GS FLX Titanium sequencing). As such, if twopolymorphisms are at a distance of, for example, >400 bp from oneanother, the likelihood of being linked by paired reads derived from alibrary is practically zero. The same is true for reads longer than 400bp as it is assumed that sequencing reads may increase in length in thefuture, however the disclosed methods would still be applicable as, iftwo polymorphisms are at a greater distance than the sequence read thecurrent methods could be utilized to determine a haplotype frompolymorphisms located on separate reads.

The present disclosure provides solutions for characterizing genomichaplotypes (e.g. haplotype content or phase) which are particularlyuseful when dealing with short read length sequence information. Thepresent disclosure provides methods and compositions for enablinghaplotype characterization from sequence information, in particular whenthe alleles of interest are located on different sequenced nucleic acidfragments.

Embodiments herein disclose methods for creating “artificialpolymorphisms” or “synthetic polymorphisms” such as artificial orsynthetic single nucleotide polymorphisms or “artificial SNPs”(“synthetic SNPs”) which can be incorporated into nucleic acids prior tosequencing such as by replacing a native nucleotide with a modifiednucleotide, or by converting one nucleotide to another through bisulfiteconversion. As used herein, the terms “synthetic polymorphism” or“artificial polymorphism”, are synonymous unless otherwise stated.Synthetic or artificial polymorphisms represent sequences in a nucleicacid sample that are not naturally occurring in the nucleic acid sample,but instead are incorporated by methodological means into the nucleicacid sample. The synthetic polymorphism could be inserted into thesequence of a genome, or the synthetic polymorphism could replace asequence of the nucleic acid sample. Examples of synthetic polymorphismsinclude, but are not limited to, single nucleotide polymorphisms (i.e.,artificial or synthetic SNPs), dinucleotide polymorphisms, insertions ofnucleic acids (e.g., one or more nucleic acids, etc.) and deletions ofnucleic acids (e.g., one or more nucleic acids, etc.). The artificialsequences for incorporation into a natural nucleic acid orpolynucleotide sample comprise modified nucleotides including, but notlimited to, 2-thio thymidine triphosphate,5-(2′-deoxy-D-ribofuranosyl)-3-methyl-2-pyridone-5′ triphosphate,8-oxoguanine (8-hydroxyguanine, 8-oxo-7,8-dihydroguanine or2-amino-7,9-dihydro-1H-purine-6,8-dione),8-Oxo-2′-deoxyguanosine-5′-triphosphate, 2′-Deoxy-P-nucleoside-5′triphosphate (dPTP), d^(5m)CTP for example, m7G(5′)ppp(5′);P1-5′-(7-Methyl)-guanosine-P3-5″-guanosine triphosphate, methyl5-dCTP,hydroxymethyl dCTP, isocytosine, isoguanine, and derivatives thereof, toname but a few.

The artificial or synthetic polymorphisms can be incorporated, forexample, at a certain frequency such that they can be aligned and phasedeven from short sequence reads or pairs of reads. In one embodiment, amethod for creating artificial polymorphisms in a nucleic acid strandcomprises incorporating a plurality of nucleic acid analogs, for examplea guanine analog such as 8-oxoguanine (8-oxo G), into a nucleic acidstrand. The amount of modified nucleotide 8-oxoguanine(8-hydroxyguanine, 8-oxo-7,8-dihydroguanine or2-amino-7,9-dihydro-1H-purine-6,8-dione (IUPAC)) found normally inmammalian DNA increases in DNA, for example that is damaged due tooxidative damage caused by oxygen free radical species and/or ionizingradiation (1992, Cheng et al., J Biol Chem 267:166-172, incorporatedherein by reference in its entirety). During replication, 8-oxo G canbase pair to either a cytosine (C) and/or adenine (A) via Hoogsteen basepairing (LePage et al., Nucl Acids Res, 1998, 26:1276-1281, incorporatedherein by reference in its entirety). The 8-oxo G (e.g., byincorporation during an extension reaction of8-Oxo-2′-deoxyguanosine-5′-triphosphate or 8OxodGTP) can be incorporatedinto a polynucleotide by a variety of means, for example by ionizingradiation or another means of oxidatively stressing the cellular DNA.Alternatively, the modified nucleotide can be added to a dNTP mix and,during an extension reaction of one or both strands of a polynucleotidecan be incorporated into an extended DNA strand thereby replacing thenormally incorporated non-modified nucleotide at a certain frequency.Following incorporation of the 8-oxo G into a strand of thepolynucleotide, adenine mispairing can be accomplished during a DNAreplication step by pairing of an adenine in the replicating strandopposite the 8-oxo G in the parent strand.

In one embodiment, 8-oxo G can be incorporated into a polynucleotideprior to library preparation for sequencing. For example, a genomic DNAsample can be fragmented, the fragment ends repaired, adenines added tothe ends via A-tailing and primer adaptors added to the ends forreplication and amplification, for example. During replication of thefragments 8OxodGTP can be added along with a canonical dNTP mix (dATP,dTTP, dGTP and dCTP) which would result in the replacement of aplurality of guanines with a plurality of 8-oxo G guanine analogs intothe DNA fragment in a random fashion. The percent of 8OxodGTP can beempirically determined. In some embodiments, the percent of 8OxodGTP isat least 10%, at least 20%, at least 30%, at least 30%, at least 50%, atleast 60%, at least 70%, at least 80% at least 90% or at least 100% ofguanines (e.g., as a replacement for dGTP) available for incorporationduring fragment replication. The percentage, and therefore ratio, ofguanine analog compared to the canonical dGTP can be empiricallydetermined for the amount of replacement desired by the user. It will beunderstood that similar percentages or ratios can be used for othernucleotides (or modified nucleotides) that are incorporated into nucleicacids using methods and compositions set forth herein, for example, inorder to introduce artificial SNPs. Continuing with the example of 8-oxoG, the genomic fragments containing 8-oxo G can be subsequently isolatedfrom those fragments that lack 8-oxo G. Isolation of the 8-oxo Gcontaining fragment can be by any means. For example, a primer usedduring replication could be complexed with a binding molecule that bindsa binding partner for isolation purposes. Such binding partner pairsinclude, but are not limited to, haptens, small molecules, dyes andantibodies such as for example biotin/streptavidin, biotin/avidin,biotin/neutravidin, DNP/anti-DNP, DIG/anti-DIG, etc. Isolation of 8-oxoG containing DNA can also be isolated by capture with an 8-oxo Gspecific antibody such as Oxoguanine 8 antibody [2Q2311] (ab64548 fromAbCam). The 8-oxo G containing DNA can also be eliminated fromdownstream haplotyping methods by either denaturation and washing ordigestion for example with formamidopyrimidine DNA glycosylase (Fpg)(also known as 8-Oxoguanine DNA glycosylase, NEB).

FIG. 1 exemplifies an embodiment using 8OxodGTP in methods forincorporating synthetic polymorphisms into genomic DNA. In FIG. 1,genomic DNA can be randomly fragmented into large fragments. The size ofthe initial large fragments can be at least 500 bp, at least 750 bp, atleast 1000 bp, at least 1500 bp, at least 2000 bp, at least 3000 bp, atleast 4000 bp, at least 5000 bp. The size of the initial fragments canbe determined empirically and may vary between different regions of thegenome that have different frequencies of guanines which would affectthe amount of downstream guanine analog incorporation. Fragmentation canbe by any means, for example sonication, Hydroshearing, nebulization,mechanical shearing and transposon methodologies, etc. The fragments canbe end repaired, A-tailed and adaptor ligated. The nucleotide 8-oxo Gcan be incorporated into a strand of the genomic fragment by primerextension and a dNTP mix that includes 8OxodGTP. The primer utilized forDNA extension and incorporation of the modified nucleotide can becomplexed with biotin which can be subsequently captured by astreptavidin molecule for isolation of the 8-oxo G containing strand.The captured 8-oxo G containing templates can be replicated resulting in8-oxo G mispairs with adenines, thereby creating double stranded DNAmolecules wherein the template contains the guanine analogs and thecopied strand contains the mispaired adenines. To remove the 8-oxo Gcontaining strands, thereby leaving the adenine containing strands, theprimer used for replication of the second strand can be affixed to acapture moiety such as biotin and capture by streptavidin can beperformed.

The remaining adenine containing polynucleotides can be furtheramplified and processed to create a library of fragments for sequencing.The created synthetic adenine SNPs in the fragments are random and, dueto the randomness of the guanine substitutions with 8-oxo G, the patternof introduced synthetic SNPs can be used to uniquely identify theparental fragments. Following sequencing the artificial SNP patterns canbe aligned among all the fragments thereby combining the fragmentsequences in the original genomic order for haplotype determination,such as determination of haplotype content or phase.

In another embodiment, a method for introducing artificial polymorphismsin a genomic DNA for sequencing comprises modifying DNA with bisulfitethereby creating a pattern of artificial polymorphisms. In one example,applying bisulfite to a nucleic acid sample in low concentration or fora short period of time can modify DNA by incompletely and partiallyconverting a subset of unmethylated cytosine residues to uracils anduracils into thymines thereafter to create artificial thyminepolymorphisms at a plurality of locations in the genomic DNA. Whenmammalian DNA is treated with bisulfite, methylated cytosines (e.g.,5-methylcytosine) remain untouched whereas cytosine residues that arenot methylated are converted to uracils. Therefore, by utilizing themethylation status of a genomic DNA sample and treating genomic DNA withbisulfite a pattern of artificial T SNPs (C to U to T) can be createdwhich can be aligned among the fragments after sequencing to reconstructthe genomic DNA chromosomal sequence for subsequent haplotypecharacterization (e.g. identification of the haplotype content orphase). In preferred embodiments, partial and incomplete conversion ofmethylated cytosine residues is preferred when practicing methodsdisclosed herein for creating a pattern of synthetic polymorphisms in apolynucleotide.

Examples of natural cytosine sequence configurations which could betargets for partial bisulfite conversion include, but are not limited toCG methylation dinucleotides (1994, Clark et al., Nucl Acids Res22:2990-2997, incorporated herein by reference in its entirety), CpT andCpA dinucleotide regions (2000, Lyko et al., Nature 408:538-540; 2000,Ramsahoye et al., Proc Nat Acad Sci 97:5237-5242; 2001, Haines et al.,Dev Biol 240:585-598, incorporated herein by reference in theirentireties) and CHG and CHH in stem cells wherein H can be either anadenine (A), cytosine (C) or thymine (T) (2009, Lister et al., Nature462:315-322, incorporated herein by reference in its entirety).

An amplification step can be utilized to create multiple copies of eachparental fragment with the newly integrated artificial SNPs prior tolibrary preparation. As such, differences between methylation patternsfound on maternal and paternal chromosomes could be exploited byfollowing the methods disclosed herein.

In other embodiments, DNA can be modified in vitro to include methylatednucleotides (e.g., modified nucleotides which are non-native methylatednucleotides). For example, methylated nucleotides can be incorporatedinto a plurality of locations in a polynucleotide by amplification, suchas amplification of a nucleic acid in the presence of canonical dNTPswherein one of the dNTPs is replaced in whole, preferentially in part,with a methylated dNTP including, but not limited to, d^(5m)CTP,m7G(5′)ppp(5′); P1-5′-(7-Methyl)-guanosine-P3-5″-guanosine triphosphate(Roche Applied Science), methyl5-dCTP (Zymo Research), or hydroxymethyldCTP (Bioline). Additionally, methylated dNTPs can be spiked into anamplification reaction in a background of canonical dNTPs. Partialbisulfite conversion could then be carried out on the in vitro modifiedDNA as described herein for creating a pattern of syntheticpolymorphisms in a nucleic acid sample.

The use of natural methylation status of a genomic DNA sample to createartificial SNPs for haplotyping and/or haplotype phasing determinationis exemplified in FIG. 2. In FIG. 2, genomic DNA is fragmented aspreviously described and the fragment ends are repaired and A-tailedusing methods known in the art (for example, see Molecular Cloning; ALaboratory Manual, Eds. Sambrook, Fritsch and Maniatus, Cold SpringHarbor Laboratory Press) as previously exemplified in FIG. 1. Theprepared genomic fragments can be ligated to adaptors for subsequentamplification of the fragments. The adaptors for use with the bisulfiteconversion method for creating artificial SNPs can be designed so thatthey are extendable and amplifiable following bisulfite treatment. Forexample, the adaptors can be pre-methylated (i.e., methylated adaptors),or adaptors could be designed which lack cytosine nucleotides whereprimer binding occurs. The adaptor ligated fragments can be amplifiedand copied using dTTP to replace the uracils prior to librarypreparation. Following library preparation and sequencing the artificialSNP patterns in the fragmented sequences can be aligned to reconstructthe original genomic DNA which can then be haplotyped. The partialconversion of cytosines by bisulfite conversion creates synthetic SNPsin the fragments wherein, due to the randomness of the conversions, thepattern of synthetic SNPs can be used to uniquely identify the parentalfragments.

Alternatively, in some embodiments the partial conversion of cytosinesto uracils can be performed prior to genomic DNA fragmentation and/oradaptor ligation, in which case the ligated adapters need not bemethylated or otherwise designed to resist bisulfite treatment ofcytosines.

In another embodiment, methods for determining haplotype of a genomicsequence comprise the use of modified nucleotides such as isoC and isoG.Isocytosine (isoC, iC) and isoguanine (isoG, iG), modified nucleotideshaving the amine and ketone groups inverted as compared to the standardcytosine and guanine nucleotides, can be misincorporated into a DNAstrand resulting in the random placement of artificial polymorphisms. Inthe case of isoC and isoG, the polymorphisms created can be copied orsequenced in later steps using the correct complementary non-naturalpartner. In this embodiment, it is advantageous to misincorporate theisoC and isoG in the initial DNA replication step and change conditionsfor subsequent amplification steps (i.e., such as those used in librarypreparation methods) to minimize or preferentially stop furthermisincorporation (2005, Sismour and Benner, Nucl Acids Res 33:5640-5646,incorporated herein by reference in its entirety) to faithfully copy thenewly formed artificial polymorphisms.

FIG. 3 is exemplary of the use of modified nucleotides in methods forcreating artificial polymorphisms in DNA. For example, genomic DNA canbe fragmented as previously described. Adaptors can be ligated to theends of the random fragments as previously described. Exemplarynaturally occurring SNPs A and T are depicted on one of the fragments;these SNPs being targeted as an example for haplotyping. Duringextension, a modified nucleotide, in this example iC, can beincorporated into the extended strand which is further end labeled witha binding moiety affixed to the extension primer, in this examplebiotin. The modified nucleotide deoxyisocytosine diCTP can be part ofthe extension dNTP mix in a defined ratio or percentage. Such ratios orpercentages can be determined empirically for the amount of syntheticpolymorphism incorporation desired by an investigator. The strandcomprising the modified nucleotide can be captured with the bindingpartner, in this case streptavidin and subsequent strand duplication canincorporate the mate to the modified nucleotide, in this case iG asdescribed for iC. The double stranded fragments, which comprise iC onone strand and iG on the other can be amplified thereby creatingmultiple fragments comprising both modified nucleotides for use inlibrary preparation.

In another embodiment, synthetic polymorphisms can alternatively beincorporated into genomic library fragments downstream of fragmentlibrary preparation. For example, once the genomic library is created(by any means known to a skilled artisan, for example as discussedherein), synthetic polymorphisms can be incorporated in steps betweenthe library preparation and sequencing. In one non-limiting example,synthetic polymorphisms can be incorporated during colony formationprior to sequence by synthesis methodologies. In this case, the DNAlibrary can be hybridized to primers affixed on a substrate and a firststrand extension reaction can be utilized to incorporate modifiednucleotides into the fragment library. This “first strand extensionreaction” format is exemplified in FIG. 6. Briefly, two primers (P1 andP2) which are homologous to primers affixed to the ends of the DNAlibrary fragments are bound to locations on a substrate such as aflowcell (e.g., lanes or wells on a flowcell), wells, plates, and thelike. The template DNA library fragments can be hybridized to thesubstrate bound primers and a complementary DNA strand can besynthesized (e.g., 1^(st) strand extension on FIG. 6) in the presence ofmodified nucleotides. Clustering, sequencing and aligning can beperformed to align the incorporated artificial polymorphisms to providea sequence useful for haplotype determination.

For all embodiments described herein for incorporating artificialpolymorphisms into genomic DNA for sequencing, libraries for sequencingcan be prepared using a method compatible with the downstream sequencinginstrument. The sequences of fragments, once determined, can be alignedon the basis of the synthetic SNPs present in the fragments and ahaplotype can be constructed and determined based on that alignment, forexample when the length of the sequence read is shorter than thedistance between the two alleles for haplotype determination.

The first sequences in FIGS. 4 A and B shows two exemplary alleles(allele 1 and 2) comprising naturally occurring polymorphisms, in thisexample SNPs, which are separated by more than 400 nucleotides (G-C inallele 1 and T-A in allele 2). As the distance between these SNPs isgreater than the average insert size of the library preparatory methodfor sequencing, phasing or haplotyping of the two SNPs would not bedeterminable using unmodified nucleotides. The second sequences in FIGS.4 A and B show the same region from exemplary alleles 1 and 2 afterpracticing a method of the present disclosure, for example practicingthe method of partial bisulfite conversion of the parental genomicfragments prior to sequencing. The two modified allelic sequencesdemonstrate an example of a unique pattern of artificial polymorphismswhich could be created by bisulfite conversion as disclosed herein.

After sequencing, the short length sequence reads would be aligned basedon the artificial polymorphisms to recreate the unique pattern for eachallele, thereby reconstructing the original genomic DNA fragment (FIG.5). The haplotype reconstruction of the two alleles, using allele 2 inFIG. 5, is determined following fragment alignment based on syntheticpolymorphic patterns. As such, incorporating synthetic polymorphismsinto a nucleic acid molecule prior to sequencing allows for a uniquesynthetic pattern which can be subsequently aligned post sequencingamong the different sequence fragments, thereby providing a means forbridging the distance between the naturally occurring SNPs to determinetheir haplotype content or phase.

Additionally, methods disclosed herein provide a means for determiningthe origin of the sequenced fragments. For example, the relativefrequency of artificial polymorphism creation and their random natureenables the determination of whether or not two DNA sequencingpopulations (e.g., two or more DNA clusters, isolated populations of DNAamplicons derived from one template, etc.) are derived from the sameoriginal parental DNA molecule. If two or more populations share thesame overlapping pattern of artificial polymorphisms, it is contemplatedthat they are derived from the same chromosome and therefore all of thenatural SNPs present in the populations can be haplotypes or phasedtogether.

Therefore, the methods of creating artificial polymorphisms in a targetgenomic sequence which are designed to occur at a much higher frequency(or in closer proximity) in the target genomic DNA compared to thefrequency (or proximity) of naturally occurring SNPs can be exploited tolink naturally occurring SNPs in a target sequence when it was notpreviously possible due to the distance of separation between thenaturally occurring SNPs in the target relative to the sequence readlength. Moreover, embodiments for creating artificial polymorphisms in atarget genomic DNA as disclosed herein require no prior knowledge of thesequence being haplotyped. Although the creation of artificialpolymorphisms does fundamentally change the sequence being evaluated, itis possible to remove the artificial polymorphisms from the finalconsensus sequence of a region by either comparing to a 2^(nd) librarywith no artificial polymorphisms, or by ignoring the artificialpositions and using sequence data from other fragments to cover thosebases (for example, an artificial polymorphism can be identified andignored if it occurs in for example 5-10% of fragments covering aparticular position).

In another embodiment, methods for determining a haplotype of a nucleicacid sample comprise incorporating artificial polymorphisms into thenucleic acid by biased amplification. Exemplary methods for performingbiased amplification can be found at, for example, WO2011/106368(incorporated herein by reference in its entirety). Biased amplification(i.e., the process of increasing the numbers of a polynucleotide whichcan be linear or exponential) may comprise amplifying the targetsequences wherein said amplification results in a deoxyribonucleotidetriphosphate (dNTP) being incorporated into the nucleic acid strand at alower efficiency compared to another nucleotide. The methods may use apool of dNTPs, wherein not all of the dNTPs (i.e., dATP, dTTP, dCTP,dGTP) are present at the same concentration in the pool. Pools ofnucleotides may also include modified nucleotides such as thosepreviously mentioned, which incorporate less efficiently (or less often)than canonical nucleotides.

For example, one or more of the dNTPs may be present at a concentrationthat is less than half of the combined concentrations of any othernucleotide in a step carried out in a method set forth herein such as anamplification reaction step. The concentration of any one type of dNTPmay be, for example, less than ¼ the concentration of the other combinednucleotides, less than ⅕ the concentration of the other combinednucleotides, less than 1/10 the concentration of the other combinednucleotides, etc. Alternatively, the concentration of a particular typeof dNTP in an amplification reaction may be less than 20 uM, less than10 uM, less than 0.2 uM compared to the concentration of the remainingdNTPs (e.g., 200 uM) present for an amplification reaction.Alternatively, the concentration of a particular type of dNTP in acomposition or method set forth herein could be at least 5 fold less, atleast 10 fold less, at least 20 fold less, at least 50 fold less thanthe concentration of the remaining dNTPs that are present. In such abiased mixture, one or more adjuvants may be added. For example,ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethylsulfoxide, glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride, salt or carboxymethyl trimethyl ammonium.Concentrations of the one or more adjuvants may be between, for example,2 to 5M. A skilled artisan will understand that conditions may vary fromreaction to reaction; as such some optimization for any particularsystem is contemplated (for example, amplification reaction conditionscan be optimized in accordance with WO2011/106368, which is incorporatedherein by reference in its entirety).

It is contemplated that incorporating the synthetic polymorphisms asdescribed herein into target nucleic acids of interest prior to librarypreparation is advantageous for a variety of reasons. For example, themethods for incorporating synthetic polynucleotides into nucleic acidsas described herein can be performed in conjunction with any librarypreparation method regardless of assay instrument (e.g., librarypreparation protocols for use in sequencing instrumentation including,but not limited to, those of Illumina, Inc., Applied Biosystems®, IonTorrent®, 454 Life Sciences, Complete Genomics, Pacific Biosciences,Oxford Nanopore Technology, etc.). Further, practicing the methodsdescribed herein upstream of library preparation protocols allows thesynthetic polymorphisms to be fixed and determinable prior to librarypreparation. Additionally, practicing the methods described hereinprovides for an initial fragmentation of genomic DNA into longerfragments, for example more than 100 bp, more than 300 bp, more than 500bp, more than 1000 bp, more than 2000 bp, more than 10,000 bp, etc.Longer fragments, while typically not advantageous for next-generationsequencing, allow for the incorporation of more synthetic polymorphismsthan would shorter fragments (e.g., <300 bp); as such providing apattern of synthetic polymorphisms which, upon additional fragmentationof longer fragments into shorter fragments, can be readily discernibleand alignable after sequencing. Another advantage of longer fragments isthat longer fragments have the possibility of containing greater thanone natural SNP as such more SNPs can be identified and aligned usingfewer fragments.

In some embodiments, synthetic nucleotides can be incorporated intonucleic acids prior to nucleic acid fragmentation. For example, modifiednucleotides could be incorporated into cellular nucleic acids duringcell culture. Modified nucleotides could be incorporated into cellularnucleic acids for example by modifying the culture media to include themodified nucleotides in a concentration sufficient to causeincorporation of the modified nucleotides into cellular DNA.

In other embodiments, genomic DNA can be rendered into smaller genomicmolecules comprising modified nucleotides without the need formechanical, chemical, or biological fragmentation following by modifiednucleotide incorporation. For example, instead of initial fragmenting ofthe genomic DNA by, for example mechanical or biological methods (e.g.transposon related methods), randomers (e.g., random sequence hexamers)could be utilized for creating a plurality of nucleic acid moleculesderived from the genomic DNA template. For example, randomers could behybridized to genomic DNA and extended (e.g., by rolling circleamplification) thereby creating long strands of DNA which would servethe same purpose of other forms of fragmentation disclosed herein (e.g.,create smaller polynucleotides for library preparation for sequencing).The extension products resulting from the extension could then be usedin bisulfite conversion methods for converting natural nucleotides tosynthetic polymorphisms. In other embodiments, modified nucleotides(e.g., pPTP, 8-oxo-G, isoC, isoG, etc.) could be incorporated during theextension reaction resulting in extension products that contain themodified nucleotides thereby concatenating the steps of creating shortermolecules from genomic DNA comprising modified nucleotides, which canthen be used for further library preparatory methods.

Regardless of method for incorporating synthetic polymorphisms intonucleic acid molecules, the resulting polynucleotides comprising thesynthetic polymorphisms can be used for downstream assays. For example,the modified nucleic acid molecules can be utilized for sequencing. Thenucleic acid molecules comprising the synthetic polymorphisms findparticular utility for determining or characterizing a haplotype of asample. The nucleic acid molecules comprising the syntheticpolymorphisms also find particular utility for de novo sequencing whereshorter sequence reads can be aligned and assembled to create fulllength, and sometimes novel, sequences. The nucleic acid moleculescomprising the synthetic polymorphisms also find particular utility whensequencing regions in the genome that comprise high incidence ofrepeated regions which can be difficult to align due to their repetitivenature.

The random nature of incorporating the synthetic polymorphisms using themethods disclosed herein provides a modified nucleic acid molecule witha pattern of incorporated polymorphisms, that random pattern of which,once determined, can be aligned and reported for determining a samplehaplotype (e.g. haplotype content or phase), a de novo sequence,verification of a sample sequence, the sequence of genomic locationsthat were previously deemed difficult to determine, etc. Sequencesdetermined by practicing methods disclosed herein, for example adetermined haplotype, can be used by diagnosticians, clinicians,researchers and other parties for example for correlating sequences todisease states (e.g., cancers, neurological disorders, degenerativedisorders, etc.) information which in turn can be utilized to diagnoseand predict whether or not an individual may or may not have, or may ormay not have a predisposition to, a particular disease or disorder.Further, certain sequences, for example a haplotype, may be correlatedto preferential treatment regimens for a particular disease or disorderwhich may be used by health care professionals to determine a treatmentregimen specific to any particular individual. Additionally, methods canbe used to determine the type and number of repeated regions in agenome, for example for forensic purposes.

In some embodiments, the modified nucleic acid molecules comprisingsynthetic polymorphisms can find particular utility in sequencing, forexample for determining a haplotype, for de novo sequencing, etc. Themodified nucleic acid molecules comprising synthetic polymorphisms canbe sequenced by any means. Target nucleic acids, for example genomicDNA, are typically extracted and isolated from a sample prior tosequencing. Alternatively, RNA may be harvested from a sample and cDNAcreated from the isolated RNA, wherein the cDNA can be used forsequencing. The terms “nucleic acid” and “polynucleotide” refer todeoxyribonucleic acid (DNA), ribonucleic acid (RNA), complementary DNA(cDNA) or analogues of DNA, cDNA or RNA. The nucleic acids can be singlestranded or double stranded molecules. The nucleic acids orpolynucleotides may have originated in single stranded form, such asssDNA or RNA, or they may have originated in double stranded form(dsDNA) such as that found in genomic DNA, amplification products,and/or fragments thereof, and the like. The nucleic acids orpolynucleotides, regardless of stranded nature, may derive from anynumber of sources including, but not limited to, a sample from an entiregenomic complement of an organism, a fragment of an entire genomiccomplement of an organism. Nucleic acids may include intronic and exonicsequences or any number of regulatory and/or non-regulatory sequences.

A sample can be from any source, for example, prokaryote, archaea oreukaryote. Further, a sample can be liquid (i.e., blood, serum, plasma,cerebral spinal fluid, urine, etc.) or solid (i.e., cells, tissues,etc.). As used herein, the term “sample” is used consistent with itsmeaning in the art of biology and chemistry. In one sense, it is meantto include a nucleic acid or polynucleotide or fragment thereof from aspecimen or culture obtained from any source such as biological andenvironmental samples. Biological samples may be obtained from animalsincluding, but not limited to humans, non-human primates, and non-humananimals including, but not limited to, vertebrates such as rodents,ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines,canines, felines, ayes, etc. Biological samples include, but are notlimited to, fluids such as blood products, tissues, cells, and the like.Biological samples can further be of plant origin, monocotyledonous ordicotyledonous, deciduous or evergreen, herbaceous or woody, includingbut not limited to agricultural plants, landscape plants, nurseryplants, and the like. Environmental samples may be bacterial, viral,fungal, and the like, in origin. Preferred samples are eukaryotic inorigin. Particularly useful samples are those derived from organismshaving more than one set of haploid chromosomes (the set being one ormore different chromosomes). For example, a sample can be derived froman organism that is diploid, triploid or polyploid. Basically, anyorganismal nucleic acid sample source of interest to an investigator indetermining sequence information is amenable to the present methods. Asample can also include a synthetic nucleic acid or fragment thereof.Derivatives or products of nucleic acids such as amplified copies orchemically modified species are also included. In preferred embodiments,a sample is derived from a mammal, for example a human.

A variety of methods and protocols are available for isolating nucleicacids (such as genomic DNA or RNA) from a sample as known to a skilledartisan, for example as described in Molecular Cloning: A LaboratoryManual (Eds., Sambrook, Fritsch and Maniatus, Cold Spring HarborLaboratory), Current Protocols in Molecular Biology, John Wiley & Sons,Inc. (The Red Book) and Short Protocols in Molecular Biology, Eds.,Ausubel et al., John Wiley & Sons, Inc. There are also a myriad ofcommercially available products and kits available for isolating DNA andRNA from a variety of sample types. The present disclosure is notintended to be limited by the way in which nucleic acids are isolatedfrom a sample.

Following nucleic acid extraction and isolation from a sample, thenucleic acids can be processed further prior to sequencing, for examplefollowing library preparation protocols. Processing may differ dependingon which sequencing instrument and technology is being utilized by theinvestigator. Methods and systems disclosed herein are not necessarilylimited to any particular library preparation method or technology.FIGS. 1-3 exemplify practicing the disclosed methods, for example insome embodiments, prior to practicing library preparation. Even thoughthere are advantages for performing the methods disclosed herein priorto typical library protocols wherein smaller fragments of genomic DNAare desired, the methods can be incorporated into the workflow of atypical library preparation methodology. For example, the methodsdisclosed herein could also be incorporated into any library preparationstep prior to sequencing of the sample. As such, in some embodiments,the methods for incorporating synthetic polymorphisms into target DNAcan be incorporated into a library workflow following libraryfragmentation of the sample and prior to sequencing the sample DNA. Asan example, the method described herein may be incorporated into, orused in combination with, the sample preparation workflow for PACBIO RSDNA Template Preparation Kit (Pacific Biosciences, Inc., Menlo Park,Calif.) which utilizes SMRTbell™ technology library format where insertlengths for sequencing can be between 250 and 6000 bp long. Aninvestigator can utilize PCR related methods for library preparation orcan alternatively employ non-PCR based methods for library preparation.

As exemplified in FIGS. 1-3, in some embodiments genomic DNA representedas a pair of homologous chromosomes can be randomly fragmented into longpieces of DNA fragments, for example fragments at least 300 bp, at least500 bp, at least 750 bp, at least 1000 bp, at least 2000 bp, at least3000 bp, at least 5000 bp long. Random fragmentation can be accomplishedby a variety of means known to a skilled artisan. For example, in someembodiments mechanical and/or acoustic shearing can be used to fragmentgenomic DNA such as by repeatedly forcing a genomic DNA sample through asmall bore syringe, by nebulization, by hydroshearing or by sonication.

Initial fragmentation of nucleic acids can be the same or different asthose utilized for a variety of library preparation protocols. Examplesof nebulization effected fragmentation of DNA is described in thePaired-End Sample preparation kits by Illumina, Inc and in kits forgenerating library DNA utilized by the GS Junior and GS FLX sequencingsystems of 454 Life Sciences (Branford, Conn.). In some embodiments,shearing of DNA is accomplished by hydrodynamic forces, for example asthose provided by the DIGILAB® HydroShear technology instruments aredescribed in the workflow for the SOLiD™ Mate Paired library kits(Applied Biosystems® Life Technologies, Carlsbad, Calif.). In someembodiments, shearing of DNA is accomplished by acoustic/mechanicalmeans such as that provided by Covaris® adaptive focused acoustics (AFA)processes. In some embodiments, sonication may also be used forfragmenting genomic DNA for example as exemplified in the workflow ofthe SOLiD™ Fragment Library construction kits (Applied Biosystems® LifeTechnologies, Carlsbad, Calif.) wherein Covaris® sonication technologyis utilized to shear genomic DNA. In some embodiments, transposon basedtechnology can be utilized for fragmenting DNA, for example asexemplified in the workflow for Nextera™ DNA sample preparation kits(Illumina, Inc.) wherein genomic DNA can be fragmented by an engineeredtransposome that simultaneously fragments and tags input DNA(“tagmentation”) thereby creating a population of fragmented nucleicacid molecules which comprise unique adapter sequences at the ends ofthe fragments. Transposon based methodologies are particularlyadvantageous when long nucleic acid fragments are desired. In someembodiments, enzymatic fragmentation can be utilized to fragment genomicDNA, for example as employed in the workflow of Ion Plus and Ion Xpress™Plus and fragment library kits (Ion Torrent™ Life Technologies,Carlsbad, Calif.). As demonstrated, there are a myriad methods forfragmenting large nucleic acid molecules, such as genomic DNA, and askilled artisan will understand that the method may be determined basedon a particular assay technology and instrument.

In some embodiments, once the nucleic acids for assay are initiallyfragmented into long fragments as previously described furtherprocessing of the sample may be performed. As exemplified in FIGS. 1-3,some embodiments comprise the affixation of additional sequences, suchas adapter sequences, on the ends of nucleic acid fragments. Adaptersequences may be used for additional downstream methods such asamplification, polymerase chain reaction, molecule capture methods, andthe like. Such adapter sequences may be primer sequences which may bethe same or different than adapter sequences utilized in downstreamlibrary preparation kits and methods. Adaptors may be double stranded,single stranded, forked (i.e., a portion of the adaptor being doublestranded and a portion of the adaptor being two single strands) or inhairpin configuration (i.e., a portion of the adaptor being doublestranded and a portion being a single stranded loop structure). Adaptorscould also include unique sequences, such as barcodes, useful inidentifying a particular target DNA. The methods disclosed herein arenot necessarily limited to any particular use or sequence of adapters,and a skilled artisan will understand that use of adapters may be chosenbased on the assay and instrument being used.

FIGS. 1-3 show exemplary embodiments for incorporation of syntheticpolymorphisms into nucleic acids. For example, as seen in FIGS. 1-3 theincorporation of a modified nucleotide (e.g., 8-oxo G), bisulfiteconversion of C to U, and incorporation of a modified nucleotide (e.g.,iC), respectively, can be performed for creating synthetic polymorphismsin nucleic acids. In some embodiments, the modified nucleotide 8-oxo Gcan be incorporated into double stranded DNA by exposing the nucleicacid fragments to oxygen free radical species and/or ionizing radiation.Alternatively, 8-oxo G can be incorporated into a nucleic acid byannealing and extension of a primer on the nucleic acid in the presenceof canonical nucleotides dATP, dTTP, dCTP and a ratio of dGTP to theanalog 8OxodGTP. In some embodiments, the ratio of dGTP to 8OxodGTP isat least 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:75,1:99. In other embodiments the percentage of 8OxodGTP in a method forincorporating synthetic polymorphisms is 100% (i.e., no dGTP is added toa reaction). The same or similar process can be followed forincorporation of modified nucleotides such as iC and iG, as exemplifiedin FIG. 3. For partial bisulfite conversion, conventional methods forbisulfite conversion known to a skilled artisan can be followed forpartial conversion of cytosines to uracils in DNA as exemplified in FIG.2.

In some embodiments, one or more primers utilized to bind to the adaptersequences for incorporation of modified nucleotides by annealing andextension of the primers may be further associated with a binding moietyfor effecting capture and purification of the modified nucleic acidstrand from the non-modified strands (i.e., nucleic acid strands with noincorporated synthetic polymorphisms). As exemplified in FIGS. 1 and 3,the hapten biotin can be associated with a primer for subsequent captureby its binding partner streptavidin, thereby purifying it away from thenon-modified nucleic acids. However, the present methods are notnecessarily limited by a particular type or set of binding partners orcapture system. In some embodiments, once the strand containing themodified nucleotide is captured and purified away from the non-modifiedstrand, the modified strand can be duplicated and syntheticpolymorphisms replicated, for example by primer binding to an adapteraffixed to the end of a nucleic acid followed by duplication to create adouble stranded nucleic acid molecule with incorporated syntheticpolymorphisms.

In some embodiments, there is no selective capture of strands. Forexample, FIG. 2 demonstrates a method for incorporating syntheticpolymorphisms wherein selective capture is not performed. Thisdemonstrates that even though strand selection is advantageous it is notalways required. In some embodiments, once a nucleic acid strandcomprising modified nucleotides is purified and/or selected from itscomplement which does not comprise modified nucleotides the selectedstrand can be replicated by, for example, primer extension methods,wherein such replication or duplication incorporates syntheticpolymorphisms opposite the location in the parent strand wherein residesthe modified nucleotides. As exemplified in FIG. 1, duplication of thetemplate nucleic acid strand comprising 8-oxo G results in acomplementary strand comprising newly incorporated adenines (A) oroccasionally cytosines (C) opposite the location of 8-oxo G nucleotidesin the template strand. However, adenines are exemplary of a nucleotidewhich mispairs with 8-oxo G. Cytosines can also pair with the modifiednucleotide 8-oxo G. As such, in some embodiments wherein 8-oxo G isutilized as the modified nucleotide for incorporating syntheticpolymorphisms, adenines and/or cytosines can be incorporated assynthetic polymorphisms. When other modified nucleotides are utilized,the resulting synthetic polymorphism being incorporated can be anucleotide which pairs with that specific modified nucleotide.

FIG. 1 demonstrates the removal of the exemplary modified nucleotide8-oxo G prior to sequencing. The nucleotide 8-oxo G can pair with eitheradenines or cytosines, as such the maintenance of the 8-oxo G in afragment for sequencing would not be preferential. In some embodiments,a modified nucleotide is maintained in nucleic acid fragments used forsequencing. For example, the incorporation of isoC (FIG. 3) into anucleic acid fragment wherein, upon duplication, the nucleotide partnerisoG is also incorporated thereby providing a nucleic acid for sequencecomprising both isoC and isoG as synthetic polymorphisms.

In embodiments of the present application, the nucleic acid fragmentscomprising the synthetic polymorphisms can be amplified. Suchamplification can enrich a library for only those nucleic acid fragmentsthat comprise adapters at both ends as well as to increase the amount ofDNA in the fragment pool going into the library preparation process. Forexample, polymerase chain reaction (PCR) amplification can be performedafter incorporation of synthetic polymorphisms into nucleic acidfragments using primers that anneal to the adapters ligated to the endsof the nucleic acid fragments. Adapters as used herein may serve manyfunctions, one of which is for hybridization to homologous sequencesaffixed to substrates, for example for performing emulsion PCR (emPCR)or clonal generation for use in sequence by synthesis methodologies.

After the target nucleic acids have been modified to comprise aplurality of synthetic polymorphisms, a library preparation forsequencing can be produced, for example, by performing the methodsrecommended by a particular sequencing method and instrument. Forexample, as described in protocols and manuals for use in any number ofsequencing systems including, but not limited to, Illumina, Inc. (e.g.,HiSeq 1000, HiSeq 2000, HiSeq 2500, MiSeq, Genome Analyzer systems,etc.), 454 Life Sciences (e.g., GS Junior, GS FLX+, etc.), AppliedBiosystems® Life Technologies (e.g., SOLiD™ sequencing systems) and IonTorrent™ Life Technologies (e.g., Ion PGM™ Sequencer, Ion Proton™Sequencer, etc.). A DNA library sample may be further amplified forsequencing by, for example, multiple strand displacement amplification(MDA) techniques. A skilled artisan will recognize additional methodsand technologies for producing nucleic acid libraries which could alsobe used in combination with methods described herein for incorporatingsynthetic polymorphisms into nucleic acid fragments. As such,embodiments described herein are not necessarily limited to anyparticular method for creating libraries, other than, in particularembodiments, the incorporation or creation of synthetic polymorphismsprior to or within those methods.

Nucleic acid libraries comprising synthetic polymorphisms areadvantageous for use in sequencing assays, for example for determininghaplotypes, de novo sequence determinations and forensic nucleotideapplications (i.e., nucleotide repeat regions, etc.) to name a few. Insome embodiments, DNA libraries comprising synthetic polymorphisms canbe immobilized on a flowcell. The immobilized nucleic acids can besequenced using single molecule resolution techniques or the immobilizednucleic acids can be amplified, for example via bridge amplification,for ensemble-based detection. Bridge amplification can be performed onthe immobilized polynucleotides prior to sequencing, for example forsequence by synthesis methodologies. In bridge amplification, animmobilized polynucleotide (e.g., from a DNA library) is hybridized toan immobilized oligonucleotide primer. The 3′ end of the immobilizedpolynucleotide molecule provides the template for apolymerase-catalyzed, template-directed elongation reaction (e.g.,primer extension) extending from the immobilized oligonucleotide primer.The resulting double-stranded product “bridges” the two primers and bothstrands are covalently attached to the support. In the next cycle,following denaturation that yields a pair of single strands (theimmobilized template and the extended-primer product) immobilized to thesolid support, both immobilized strands can serve as templates for newprimer extension. Thus, the first and second portions can be amplifiedto produce a plurality of clusters in a process known as “clustering”.Clusters and colonies are used interchangeably and refer to a pluralityof copies of a nucleic acid sequence and/or complements thereof attachedto a surface. Typically, the cluster comprises a plurality of copies ofa nucleic acid sequence and/or complements thereof, attached via their5′ termini to the surface. Exemplary bridge amplification and clusteringmethodology are described, for example, in PCT Patent Publ. Nos.WO00/18957 and WO98/44151, U.S. Pat. No. 5,641,658; U.S. Patent Publ.No. 2002/0055100; U.S. Pat. No. 7,115,400; U.S. Patent Publ. No.2004/0096853; U.S. Patent Publ. No. 2005/0100900, U.S. Patent Publ. No.2004/0002090; U.S. Patent Publ. No. 2007/0128624; and U.S. Patent Publ.No. 2008/0009420, each of which is incorporated herein by reference inits entirety. The compositions and methods as described herein areparticularly useful in sequence by synthesis methodologies utilizing aflowcell comprising clusters.

Emulsion PCR (emPCR) methods for amplifying nucleic acids prior tosequencing can also be used in combination with methods and compositionsas described herein. Emulsion PCR comprises PCR amplification of anadaptor flanked shotgun DNA library in a water-in-oil emulsion. The PCRis multi-template PCR; in particular embodiments only a single primerpair is used. One of the PCR primers is tethered to the surface (5′attached) of microscale beads. A low template concentration results inmost bead-containing emulsion microvesicles having zero or one templatemolecule present. In productive emulsion microvesicles (an emulsionmicrovesicle where both a bead and template molecule are present), PCRamplicons can be captured to the surface of the bead. After breaking theemulsion, beads bearing amplification products can be selectivelyenriched. Each clonally amplified bead will bear on its surface PCRproducts corresponding to amplification of a single molecule from thetemplate library. The beads can then be arrayed on a surface of a flowcell for sequencing. Various embodiments of emulsion PCR methods are setforth in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822(2003), PCT Patent Publ. No. WO 05/010145, U.S. Patent Publ. Nos.2005/0130173, 2005/0064460, and 2005/0042648, each of which isincorporated herein by reference in its entirety.

DNA nanoballs can also be used in combination with methods andcompositions as described herein. Methods for creating and utilizing DNAnanoballs for genomic sequencing can be found at, for example, U.S. Pat.Nos. and publications U.S. Pat. No. 7,910,354, 2009/0264299,2009/0011943, 2009/0005252, 2009/0155781, 2009/0118488 and as describedin, for example, Drmanac et al., 2010, Science 327(5961): 78-81; all ofwhich are incorporated herein by reference in their entireties. Briefly,following genomic library DNA fragmentation adaptors are ligated to thefragments, the adapter ligated fragments are circularized by ligationwith a circle ligase and rolling circle amplification is carried out (asdescribed in Lizardi et al., 1998. Nat. Genet. 19:225-232 and US2007/0099208 A1, each of which is incorporated herein by reference inits entirety). The extended concatameric structure of the ampliconspromotes coiling thereby creating compact DNA nanoballs. The DNAnanoballs can be captured on substrates, preferably to create an orderedor patterned array such that distance between each nanoball ismaintained thereby allowing sequencing of the separate DNA nanoballs. Insome embodiments such as those used by Complete Genomics (Mountain View,Calif.), consecutive rounds of adapter ligation, amplification anddigestion are carried out prior to circularization to produce head totail constructs having several genomic DNA fragments separated byadapter sequences.

Disclosed methods for determining a haplotype, de novo sequence, etc. byincorporation of synthetic polymorphisms into a polynucleotide orfragment thereof find particular utility when used in sequencing, forexample next generation (“nexgen”) sequencing by synthesis (SBS)technologies. Sequencing by synthesis generally comprises sequentialaddition of one or more nucleotides to a growing polynucleotide chain inthe 5′ to 3′ direction using a polymerase. The extended polynucleotidechain is complementary to the nucleic acid template affixed on thesubstrate (e.g., flowcell, chip, slide, etc.); the target sequencecomprising the synthetic polymorphism.

Disclosed method for determining haplotype, de novo sequence, etc. byincorporation of synthetic polymorphisms into a polynucleotide orfragment thereof also find utility when used in sequencing by ligation,sequencing by hybridization, and other sequencing technologies. Anexemplary sequence by ligation methodology is di-base encoding (e.g.,color space sequencing) utilized by Applied Biosystems' SOLiD™sequencing system (Voelkerding et al., 2009, Clin Chem 55:641-658;incorporated herein by reference in its entirety). Sequence byhybridization comprises the use of an array of short sequences ofnucleotide probes to which is added fragmented, labeled target DNA(Drmanac et al., 2002, Adv Biochem Eng Biotechnol 77:75-101; Lizardi etal., 2008, Nat Biotech 26:649-650, U.S. Pat. No. 7,071,324; incorporatedherein by reference in their entireties). Further improvements tosequence by hybridization can be found at, for example, US patentapplication publications 2007/0178516, 2010/0063264 and 2006/0287833(incorporated herein by reference in their entireties). Sequencingapproaches which combine hybridization and ligation biochemistries havebeen developed and commercialized, such as the genomic sequencingtechnology practiced by Complete Genomics, Mountain View, Calif. Forexample, combinatorial probe-anchor ligation, or cPAL™ (Drmanac et al.,2010, Science 327(5961): 78-81) utilizes ligation biochemistry whileexploiting advantages of sequence by hybridization. The methods forhaplotyping, de novo sequencing, etc. disclosed herein could be utilizedin combinatorial probe-anchor ligation sequencing technologies. It iscontemplated that the methods as described herein for use of syntheticpolymorphisms to determine haplotype, de novo sequence, etc. are notlimited by any particular sequencing methodology. Additional sequencingtechnologies include, but are not limited to, those practiced by one ormore of polony sequencing technology (Dover Systems), sequencing byhybridization fluorescent platforms (Complete Genomics) and sTOPtechnology (Industrial Technology Research Institute).

Single molecule sequencing can also be used with methods as disclosedherein. For example, non-amplified DNA libraries for sequencing can beprepared as previously described. The library fragments can behybridized and captured on a substrate such as a flow cell and assayedon, for example, a HeliScope™ Single Molecule Sequence instrument.Further description of single molecule sequencing can be found at, forexample, Puchkarev et al. (2009, Nat. Biotechnol. 27:847-52,incorporated herein by reference in its entirety) and Thompson andSteinmann (2010, Curr. Prot. Mol. Biol. Cpt 7, Unit 7.10, incorporatedherein by reference in its entirety).

The methods set forth herein can be used in combination with nucleicacid detection systems such as those provided by Illumina®, Inc. (HiSeq1000, HiSeq 2000, HiSeq 2500, Genome Analyzers, MiSeq, HiScan, iScan,BeadExpress systems), Applied Biosystems™ Life Technologies (ABI PRISM®Sequence detection systems, SOLiD™ System), Ion Torrent™ LifeTechnologies (Ion PGM™, Ion Proton™) 454 Life Sciences (GS Junior, GSFLX+), PacBio RS (Pacific Biosciences®), Oxford Nanopore Technologies®(GridION, MinION) or other sequencing instruments, further as thosedescribed in, for example, U.S. Pat. Nos. and patent applications U.S.Pat. Nos. 5,888,737, 6,175,002, 5,695,934, 6,140,489, 5,863,722,2007/007991, 2009/0247414, 2010/0111768 and PCT applicationWO2007/123744, and U.S. patent application Ser. Nos. 61/431,425,61/431,440, 61/431,439, 61/431,429, 61/438,486 each of which isincorporated herein by reference in its entirety.

Output from a sequencing instrument can be of any sort. For example,some current technologies utilize a light generating readable output,such as fluorescence or luminescence. Other technologies utilizesemiconductors which detect ion release and digitally output sequencebased on hydrogen ions released during incorporation of nucleotidesduring sequencing. However, the present methods are not limited to thetype of readable output as long as differences in output signal for aparticular sequence of interest is potentially determinable.

Examples of analysis software that may be used, or modified, tocharacterize output derived from practicing methods as described hereininclude, but are not limited to, Pipeline, CASAVA and GenomeStudio dataanalysis software (Illumina®, Inc.), SOLiD™, DNASTAR® SeqMan® NGen® andPartek® Genomics Suite™ data analysis software (Life Technologies),Feature Extraction and Agilent Genomics Workbench data analysis software(Agilent Technologies), Genotyping Console™, Chromosome Analysis Suitedata analysis software (Affymetrix®). It is contemplated that one ormore software programs for use with methods and compositions disclosedherein will have the capacity to recognize the incorporated syntheticpolymorphism patterns present in the fragment sequence data, align thepolymorphisms identified in the fragment sequence data and output asequence based on that alignment. In some embodiments, the output maycomprise a haplotype (e.g. haplotype content or phase) for the targetsample. In other embodiments, the output may comprise de novo sequenceinformation for the target sample. In other embodiments, output maycomprise forensic nucleotide repeat information, such a type (i.e.,sequence of repeat, location of repeat, number of short or intermediatetandem repeats, etc.

In some embodiments, sequence analysis and alignment comprises aligningthe sequence reads against a reference genome, or de novo assembly ofalignable regions, for example by barcoding introduced into the libraryfragments for sequencing as known to a skilled artisan. Depending on thedensity of the artificial SNPs, it is contemplated that standardalignment software tools could be used. For example, if synthetic SNPdensity is high, then alignment programs could be modified such thatalignments are adequately permissive enough to place sequence reads. Asan example, existing modified alignment pipelines for bisulfitesequencing could be used when synthetic SNPs are incorporated bybisulfite conversion methodologies (e.g., as described atwww.bioinformatics.babraham.ac.uk/projects/bismark). For de novoassembly, it is contemplated that built-in error correction modules canbe disabled for standard short read assemblers when reading sequencederived from practicing methods disclosed herein (2008, Zerbino andBirney, 2008, Genome Res 18:821-829, incorporated herein by reference inits entirety).

Algorithms for building haplotype blocks from short-sequence reads couldbe used with methods disclosed herein (Bansal and Bafna, 2008,Bioinformatics 24:i153-i159). Such algorithms may, however, be modifiedaway from the standard assumption of two discrete haplotypes as would beexpected when sequencing a normal diploid human DNA molecule. Forexample, the introduced synthetic SNPs would result in a larger numberof apparent or artificial haplotypes corresponding to each originalsequence fragment and therefore modifications would be made in thealgorithms to accommodate this non-standard information.

The synthetic SNPs could be identified from normal nucleotide sequencesin a number of ways. For example, the original sequence which has notbeen modified could serve as the reference sequence and therefore as thecontrol without the synthetic SNPs. In this method, the polymorphismsthat are not present in the original sequence could be identified andcorrelated with those locations in the modified sequence, therebyidentifying the locations in the modified sequence where synthetic SNPswere incorporated. Alignment could then take place using thoseidentified modified nucleotides. For consensus calling, the syntheticpolymorphisms would be expected to be unique to the original sequence.As such, by sequencing original fragments at a particular genomicposition, the frequency of the polymorphisms across the synthetichaplotypes could be estimated and compared to the expected frequency ina normal diploid human sample.

In some embodiments, the merging of artificial haplotypes can beperformed by algorithms which are modified to identify the syntheticpolymorphisms, such as HapCUT or modifications thereto (2009, Bansal andBafna). The algorithms could be modified to merge SNPs identified asnon-synthetic SNPs but derived from different synthetic haplotypes,thereby creating the true underlying haplotype aligned map.

In some embodiments, output from aligned sequences comprising bothnatural and synthetic polymorphisms could include both the locations ofthe natural polymorphisms and the locations of the syntheticpolymorphisms in the reconstructed haplotype. Alternatively, outputcould include just the natural polymorphisms in the reconstructedhaplotypes with the synthetic polymorphisms being screened out.Visualization can be accomplished in a number of ways, for example astandard genome browser such as an integrative genomics viewer (IGV)could be utilized (2011, Robinson et al., Nat Biotech 29:24-26,incorporated herein by reference in its entirety). The reconstructedhaplotypes could be annotated in the genome browser to highlight thepositions of the true, natural polymorphisms and/or the syntheticpolymorphisms (e.g., if present in the output). However, othervisualization tools may also be used as known to a skilled artisan. Thepresent methods are not necessarily limited to the algorithms, methodsor systems used for aligning and outputting or visualizing the sequencesderived from practicing the methods disclosed herein.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the disclosedmethods and compositions and are not to be construed as limiting thescope thereof.

Prior to library preparation the genomic DNA can be modified to includeartificial polymorphisms. The genomic DNA can be initially fragmentedinto large pieces (for example several kilobases). The larger fragmentsize maximizes the occurrence of two or more artificial SNPs in the samefragment while maximizing the occurrence of more heterozygous SNPs.Transposon mediated fragmentation of nucleic acids and hydroshearing areexamples of methods for generating initial DNA fragments of, forexample, between 1,000-40,000 bp.

Example 1—Synthetic Polymorphism Incorporation into phiX Genome

Sequencing experiments were performed to assess the frequency ofincorporation of modified nucleotides into a DNA strand for downstreamsequencing. A bacteriophage reference genome, phi X 174 or phiX was usedas phiX has a small, well defined genomic sequence of 5386 bases. Thetwo modified nucleosides, 8OxodGTP and dPTP, were incorporated indifferent combinations with normal dNTPs. dPTP can base-pair to both Aand G whereas 8OxoG can base pair to both A and C.

A standard paired end Illumina flow cell was seeded with a standard phiXlibrary at a concentration of 2 pM following manufacturer's protocols.Following hybridization of the library to the flowcell boundoligonucleotides, DNA molecules were copied in the flowcell lanes usingthe first strand extension method by incubating the flow cell at 40° C.for 1 hour in the presence of a DNA polymerase and various nucleotidemixes (natural and unnatural) as found in Table 1.

TABLE 1 phiX first extension assay with deoxynucleoside concentrationsLane 1 [dATP] [dCTP] [dGTP] [dTTP] [8oxo-dGTP] [dPTP] 1 100 μM 100 μM100 μM 100 μM 2 100 μM 100 μM  10 μM 100 μM 3 100 μM 100 μM 100 μM 4 100μM  10 μM 100 μM  10 μM  90 μM 5 100 μM 100 μM 100 μM  6* 100 μM  10 μM 10 μM  10 μM  90 μM  90 μM 7 100 μM 100 μM 100 μM 8 100 μM  10 μM 100μM  10 μM  90 μM *μM concentrations were rounded up to the nearest wholenumber

Following the first extension reaction (exemplified in FIG. 6), singlemolecules were clonally amplified by 35 cycles of isothermalamplification following manufacturer's protocols to yield amplificationclusters which were sequenced on an Illumina Genome Analyzer. Reads were100 cycles and data was analyzed using the standard system softwarefollowed by alignment to the phi X reference sequence using PhageAlignsystem software for paired end analysis.

Table 2 shows a summary of a sequencing run for each lane of theflowcell. Lane 1 is the control lane and is representative of sequencingoutput from a normal sequencing run using normal dNTPs. Lanes 2-6 showsequencing run output when one or both modified nucleotides areincorporated in combination with, or replacing, normal dNTPs duringfirst strand extension (dNTP concentrations from Table 1). The % ErrorRate (PF) reports the percentage of called bases in aligned reads thatdo not match up with the reference genome which, in this experiment, isreflective of the incorporation of the modified nucleotides into thetarget phiX DNA. As seen in Table 2, all the lanes where reactionconditions incorporated modified nucleotides into the phiX DNA showed ahigher error rate than the normal control (Lane 1).

TABLE 2 Sequencing run for phiX modified DNA Lane Info Tile Mean +/− SDfor Lane Lane Yield Clusters 1st Cycle % intensity after 20 Lane(kbases) (raw) Clusters (PF) Int (PF) cycles (PF) 1 92637 114410 +/−5332   91720 +/− 36408 361 +/− 9  70.08 +/− 31.37 2 56566  83912 +/−14512  56006 +/− 24144 338 +/− 17 67.14 +/− 32.57 3 45253  75960 +/−13622  49783 +/− 10991 295 +/− 37 51.47 +/− 13.47 4 90758 122472 +/−4426  99844 +/− 5727 290 +/− 29 73.47 +/− 15.73 5 6402 24030 +/− 7123 6338 +/− 4425  430 +/− 294 65.25 +/− 37.13 6 105230 133723 +/− 29330104188 +/− 36207 284 +/− 68 93.66 +/− 51.01 7 56525  70834 +/− 17637 55965 +/− 14949 292 +/− 16 71.40 +/− 5.23  8 122200 142677 +/− 29937120990 +/− 24005 344 +/− 37 85.12 +/− 2.47  Lane Info Tile Mean +/− SDfor Lane Lane Yield % PF % Align Alignment Score % Error Lane (kbases)Clusters (PF) (PF) Rate (PF) 1 92637  80.44 +/− 32.39 100.00 +/− 0.32247727.68 +/− 728715.67  3.05 +/− 5.52 2 56566  70.95 +/− 33.52 100.00+/− 0.32 10552.33 +/− 5267.76  4.45 +/− 2.98 3 45253 65.80 +/− 9.74100.00 +/− 0.27 3894.09 +/− 1202.16  27.22 +/− 3.36  4 90758 81.55 +/−4.15 100.00 +/− 0.15 8408.26 +/− 1141.61  7.78 +/− 2.19 5 6402  30.89+/− 22.39  99.93 +/− 0.17 965602.29 +/− 1247823.10 26.79 +/− 2.94  6105230  77.47 +/− 20.35 100.00 +/− 0.00 8615.87 +/− 627.55  7.17 +/−0.18 7 56525 78.71 +/− 1.89 100.00 +/− 0.00 5619.21 +/− 388.95  24.94+/− 0.13  8 122200 84.96 +/− 1.10 100.00 +/− 0.00 9267.62 +/− 149.03 6.71 +/− 0.14

FIG. 7 shows graphs of cycle versus error rates for the control (A) lanecompared to Lanes 2 (B), 3 (C) and 4(D) (lane 6 results were basicallythe same as lane 4). As demonstrated in FIG. 7, the lanes wherein firststrand extension incorporated modified nucleotides (Lanes 2, 3 and 4)show elevated error rates in comparison to the control Lane 1. Furtherthe error rates do not increase but remain constant as such it appearsthat additional synthetic nucleotides are not incorporated after thefirst extension reaction, thereby removing the potential variable foraccurate sequencing determination due to unexpected synthetic nucleotideincorporation during cluster formation and subsequent sequencing.

Sequencing data from phiX unmodified DNA showed that the majority ofsequence reads had minimal or no sequencing errors, whereas thosesequence reads derived from first strand extension reactionsincorporating modified nucleotides had a high number of errors. FIG. 8shows that incorporating the modified nucleotides into first strandextension resulted in a large number of sequenced fragments containing1, 2, 3, 4 or more synthetic SNPs relative to the control, which wouldallow for fragment alignment of synthetic SNPs and hence haplotypedetermination. It was further determined which types and frequencies ofsynthetic SNPs resulted from the different combinations of natural andmodified as found in Table 1.

FIG. 9 shows a lane by lane comparison of the mutations resulting fromthe use of dPTP during incorporation and prevalence (error rate) in thesequencing reads. As previously stated, dPTP can base-pair to both A andG thereby allowing for the following mutations to occur when dPTP isincorporated into the first strand extension product; A→G, G→A, T→C andC→T. When dPTP is incorporated in the absence of dCTP and dTTP (lanes 3and 7 in FIG. 9) the G→A mutation dominates over other types ofmutations. Conversely, when small amounts of dCTP and dTTP are presentduring the incorporation reaction (lanes 4, 6 and 8) that mutationaldomination is minimal.

Additionally, the distribution pattern of the incorporated artificialSNPs was evaluated. As demonstrated in FIG. 10, incorporation of both8OxoG (lane 2) and dPTP (lanes 3 and 4) was uniform over the entiregenome. The spike in the figures is artifactual and does not represent adisproportionate amount of synthetic SNPs at this location.

It is contemplated that reaction conditions for Lane 5 were too extreme,resulting in sequencing failure for this lane.

Example 2—Synthetic Nucleotide Incorporation into p53 Gene

A region of the p53 gene was further sequenced using PTP modifiednucleotide inserted into the gene prior to sequencing. A region of thep53 gene was amplified using oligonucleotides TP53 Exon1 3.1F(Tail-GAAACTTTCCACTTGATAAGAGGTC) and TP53 Exon 4 8.1R(Tail-GCCCCTGTCATCTTCTGTCC). The PCR mix consisted of 1× Thermopolbuffer, 26 U/ml of Taq DNA polymerase, 0.52 μM of each oligonucleotide.Reaction 1 contained 200 μM of each natural nucleotide (dATP, dCTP,dGTP, dTTP). Reaction 2 contained approximately 200 uM of dATP and dGTP,198 μM of dCTP and dTTP and 2 μM of dPTP. Reaction 3 containedapproximately 200 μM of dATP and dGTP, 180 μM of dCTP and dTTP and 20 μMof dPTP. Amplification was carried out using the following conditions:94° C. for 3 minutes followed by 38 cycles of 94° C. for 30 seconds, 50°C. for 30 seconds, 72° C. for 5 minutes. After cycling, samples wereincubated at 72° C. for 5 minutes and the temperature was lowered to 4°C. The p53 target template was an aliquot of a PCR product amplifiedfrom sample NA18507 (human 1) using Phusion polymerase in a master mix(1× final concentration). A negative control (no template) was alsoincluded.

PCR reactions 1 and 3 were loaded onto a SYBR® Safe pre-stained 1%agarose gel in TAE and the gel bands of the expected size were excisedusing the QIAQuick Gel extraction kit following manufacturer's protocol.DNA was eluted in 30 μl of Elution Buffer. A second round ofamplification was performed with Phusion polymerase in HiFi buffer withthe primers previously described. One μl of the previous eluted DNA wasused as template for the second PCR reaction (100 μl total volume). PCRconditions were as follows: 98° C. for 1 minute followed by 38 cycles of98° C. for 10 seconds, 50° C. for 30 seconds, 72° C. for 5 minutes.After cycling, samples were incubated at 72° C. for 5 minutes and storedat 4° C. PCR reactions were loaded onto a SYBR® Safe pre-stained 1%agarose gel in TAE and the DNA bands of the expected size were excisedusing a QIAQuick Gel extraction kit. DNA was eluted in 30 μl of EB.

Eluted DNA was A-tailed at 74° C. for 30 minutes with dATP and Taq in 1×Thermopol buffer in a total volume of 10 μl per sample followingstandard protocols. A 3.5 μl aliquot of A-tailed DNA was ligated intopGEM®-T Easy vector (Promega) using Quick ligase (New England Biolabs).Ligations were transformed into XL10 Gold competent cells (Stratagene).After an overnight incubation at 37° C. on antibiotic containing agarplates, single colonies were picked and inoculated into Luria Broth.Plasmid DNA was prepared from approximately 3 ml of bacterial culturefrom each clone using a QIAprep Spin Miniprep kit (QIAGEN). Plasmid DNAwas eluted in 50 μl of EB. Clones were screened for the presence of theinsert by restriction enzyme digestion with EcoRI. Positive clones(three clones from the PCR with natural dNTPs and 6 clones from the PCRin the presence of dPTP) were sequenced by capillary sequencing with theSP6 and T7 primers homologous to pGEM®-T Easy vector sequences and alsowith an internal primer specific to the p53 sequence inserts forverification of modified nucleotide incorporation.

FIG. 11 shows the SBS sequencing results from three random clones A, Band D. The sequences represent sequence runs from a region of a p53genedemonstrating natural SNPs interspersed with incorporated syntheticSNPs. The approximate locations of the natural heterozygous SNPs arerepresented by stars on the graphs. The vertical lines representlocations of SNPs and demonstrate the random and spatially distributednature of the synthetic SNP incorporation.

Based on sequencing data, it was determined that naturally occurringSNPs were correctly identified and aligned in a sequenced section fromthe p53 gene with an average sequence read length of approximately 800bp of determinable sequence.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modifications and variationsof the described methods and compositions of the present disclosure willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention.

A number of embodiments have been described. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described methods as disclosed herein that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

What is claimed is:
 1. A method for determining the sequence of anucleic acid sample comprising: a) providing a first plurality ofnucleic acid fragments having a first length, wherein said pluralitycomprises parental chromosome fragments of maternally and paternallyinherited chromosomes from a diploid, triploid or polyploid organism,wherein individual fragments in said first plurality comprise naturallyoccurring polymorphisms that form a haplotype and a plurality ofsynthetic polymorphisms, the plurality of synthetic polymorphismsforming different patterns on individual fragments of maternallyinherited chromosomes relative to corresponding paternally inheritedchromosomes and to other corresponding fragments of the maternallyinherited chromosomes, such that individual fragments from an individualmaternally inherited chromosome having an individual pattern of thedifferent patterns are distinguishable from individual fragments of acorresponding individual paternally inherited chromosome and from theother corresponding fragments of the maternally inherited chromosomesthat do not have the individual pattern and wherein the plurality ofsynthetic polymorphisms forming the different patterns are formed bymodifying the nucleic acids by partial and incomplete bisulfateconversion of cytosines in the plurality of nucleic acid fragments; b)preparing a nucleic acid library from said first plurality of nucleicacid fragments, wherein said nucleic acid library comprises a secondplurality of nucleic acid fragments of a second length less than that ofsaid first length, wherein naturally occurring polymorphisms of ahaplotype are separated on different nucleic acid fragments of saidsecond plurality, wherein each of said nucleic acid fragments of saidsecond plurality has a first pattern of said plurality of syntheticpolymorphisms that is the same as a second pattern of said plurality ofsynthetic polymorphisms present in another nucleic acid fragment of saidsecond plurality of nucleic acid fragments, wherein the first patternand the second pattern are from only one of the individual maternallyinherited chromosome or the corresponding individual paternallyinherited chromosome and the different patterns comprise the firstpattern and the second pattern, c) sequencing nucleic acid fragments ofsaid nucleic acid library, d) aligning the plurality of syntheticpolymorphisms among the nucleic acid fragments that are sequenced usingsaid first pattern and said second pattern to determine the sequence ofthe nucleic acid sample, wherein said sequence comprises said naturallyoccurring polymorphisms of said haplotype, and (e) determining that thesequence includes the naturally occurring polymorphisms of saidhaplotype on one of the individual maternally inherited chromosome orthe corresponding individual paternally inherited chromosome.
 2. Themethod of claim 1, wherein said aligning comprises matching additionalpatterns of synthetic polymorphisms of the plurality of nucleic acidfragment sequences thereby creating a sequence alignment based on theplurality of synthetic polymorphisms in the second plurality of nucleicacid fragments.
 3. The method of claim 1, wherein said sequencing is amethod selected from the group consisting of sequencing by synthesis,sequencing by hybridization, sequencing by ligation, single moleculesequencing, nanopore sequencing, and pyrosequencing.
 4. The method ofclaim 1, wherein said sequencing comprises fluorescence detection. 5.The method of claim 2, wherein said matching is performed by a computerimplemented method.
 6. The method of claim 1, wherein said sequencingcomprises sequencing by synthesis.
 7. The method of claim 1, whereinsaid sequencing comprises sequencing by hybridization.
 8. The method ofclaim 1, wherein said sequencing comprises sequencing by ligation. 9.The method of claim 1, wherein said sequencing comprises nanoporesequencing.
 10. The method of claim 1, wherein said sequencing comprisespyrosequencing.
 11. The method of claim 1, comprising determining thatthe naturally occurring polymorphisms of the haplotype are located onthe individual maternally inherited chromosome or the correspondingindividual paternally inherited chromosome.
 12. The method of claim 1,comprising determining that a first of the naturally occurringpolymorphisms of the haplotype is located on the individual maternallyinherited chromosome and a second set of the naturally occurringpolymorphisms of the haplotype is located on the correspondingindividual paternally inherited chromosome.
 13. The method of claim 1,wherein step a) comprises modifying a first plurality of nucleic acidfragments of a first length to incorporate modified nucleotides therebyproviding the first plurality of nucleic acid fragments having the firstlength, wherein said plurality comprises parental chromosome fragmentsof maternally and paternally inherited chromosomes from the diploid,triploid or polyploid organism, wherein individual fragments in saidfirst plurality comprise naturally occurring polymorphisms that form thehaplotype and the plurality of synthetic polymorphisms, the plurality ofsynthetic polymorphisms forming different patterns on maternallyinherited chromosomes relative to corresponding paternally inheritedchromosomes, such that the individual maternally inherited chromosome isdistinguishable from the corresponding individual paternally inheritedchromosome based on the different patterns.
 14. The method of claim 13,wherein the modifying comprises incorporating a mixture of unmodifiednucleotides and the modified nucleotides into the first plurality ofnucleic acid fragments during an extension reaction.
 15. The method ofclaim 1, wherein said naturally occurring polymorphisms that form saidhaplotype are at a distance on the nucleic acids of said first pluralitythat is greater than said second length.
 16. The method of claim 1,wherein said sequencing has a read length and wherein said naturallyoccurring polymorphisms that form said haplotype are at a distance onthe nucleic acids of said first plurality that is greater than said readlength.
 17. The method of claim 1, further comprising a step ofamplifying the first plurality of nucleic acid fragments prior to stepb).
 18. The method of claim 1, wherein the plurality of syntheticpolymorphisms are present in greater frequency than the naturallyoccurring polymorphisms in the first plurality.
 19. The method of claim17, wherein the amplifying results in an enrichment of nucleic acidfragments of the second length within the nucleic acid library thatinclude the first pattern or the second pattern.
 20. A method fordetermining the sequence of a nucleic acid sample comprising: a)providing a first plurality of nucleic acid fragments having a firstlength, wherein said plurality comprises parental chromosome fragmentsof maternally and paternally inherited chromosomes from a diploid,triploid or polyploid organism, wherein individual fragments in saidfirst plurality comprise naturally occurring polymorphisms that form ahaplotype and a plurality of synthetic polymorphisms introduced into theindividual fragments of the first plurality such that different patternsof the synthetic polymorphisms are formed on the individual fragments ofthe first plurality of nucleic acid fragments relative to one anotherand such that individual fragments from an individual maternallyinherited chromosome having an individual pattern of the differentpatterns are distinguishable from individual fragments of acorresponding individual paternally inherited chromosome and from theother corresponding fragments of the maternally inherited chromosomesthat do not have the individual pattern; b) preparing a nucleic acidlibrary from the first plurality of nucleic acid fragments to generate asecond plurality of nucleic acid fragments of a second length less thanthat of the first length such that the naturally occurring polymorphismsof the haplotype are separated on different nucleic acid fragments ofthe second plurality, and wherein the different nucleic acid fragmentseach comprise a same individual pattern of the different patterns on anoverlapping portion of the different nucleic acid fragments, c)sequencing nucleic acid fragments of said nucleic acid library, d)aligning the overlapping portion comprising the same individual patternamong the different nucleic acid fragments that are sequenced todetermine the sequence of the nucleic acid sample, wherein the sequencecomprises the naturally occurring polymorphisms of the haplotype, and(e) determining that the sequence including the naturally occurringpolymorphisms of the haplotype is from only one of an individualmaternally inherited chromosome or a corresponding individual paternallyinherited chromosome.